We have investigated the molecular mechanism by which Toll-Like Receptor 3 (TLR3) recognizes double stranded RNA (dsRNA). In 2005, we determined the molecular structure of the human TLR3 ectodomain. Since then, several models of TLR3 complexed to dsRNA were suggested in the literature, based on biochemical and molecular biology results. All models share the common feature: TLR3 is suggested to form a dimer upon dsRNA binding. Mouse TLR3 has been used in most experiments instead of human TLR3. The mTLR3-ECD fused to an N-terminal GP67 secretion signal sequence and a C-terminal TEV cleavage site followed by 6xHis and Strep-II tags was inserted into a baculovirus expression system by the Protein Expression Laboratory (Science Applications International Corp., Frederick, MD) and expressed in High Five cells. The expression temperature and insect cell culture media were optimized, yielding a high expression of 6mg/l for mTLR3-ECD. The crystals obtained diffracted to 2.7A resolution and the crystal structure solved by molecular replacement revealed close homology with the human TLR3-ECD. Using dsRNA molecules of different sizes, both in vivo and in vitro experiments gave the same minimum size dsRNA - approximately 45 base pairs - which can bind and activate TLR3 for downstream signaling. A mTLR3-ECD: dsRNA complex was generated, and fairly small crystals (0.04x0.04x0.02mm) were grown that diffracted to 3.4A resolution. The structure was solved by molecular replacement followed by extensive density modification before a full model could be built into the density. The crystal structure showed a dsRNA in the form of a straight helix making contact with two TLR3 ectodomains at two separate regions on each ectodomain. These contact sites were confirmed by further mutagenesis studies. TLR3 contacts the sugar phosphate backbones of the RNA and makes no contact with the bases, thus accounting for the absence of sequence specificity. There are additional interactions between the two C-terminal capping regions to form the dimer. These contacts bring the C-termini of the ectodomain close, inducing the dimerization of the cytoplasmic TIR domains, which is believed to initiate a downstream signaling cascade. Dimerization of the cytoplasmic TIR domains creates the signaling platform for adaptor recruitment. The adaptor protein used by TLR3 signaling is TRIF (TIR-domain-containing adaptor protein inducing IFN). It is hypothesized that TRIF recruitment depends on homotypic TIR domain interactions. To address the initiation mechanism for cytoplasmic TLR3 signaling, we are: 1) Producing and attempting to crystallize the TLR3-TIR domain (TIR3), determine its structure by X-ray analysis, and localize the TIR3 dimerization interface. 2) Expressing TRIF, and attempting to crystallize and determine the structure of the TIR3-TRIF complex, thus characterizing the interactions between them. TLR22 occurs exclusively in aquatic animals. TLR3 resides in the endoplasmic reticulum and recognizes relatively short dsRNA, whereas TLR22 recognizes longer dsRNA on the cell surface. TLR22 may be a functional substitute of human cell-surface TLR3 and serve as surveillance for infection with dsRNA virus, for antiviral protection in fish. Our goal is: 3) Expression, purification and crystallization of TLR22, 4) Determination of the TLR22 structure by X-ray crystallography to be able to compare it to TLR3, and 5) Determination of the structure of TLR22-dsRNA, to understand the different mechanisms of TLR recognition of dsRNA.